Lung cancer is the main cause of preventable cancer death in the Western world both in men and in women. It has been estimated that about 1 in 12 life-time smokers will eventually develop one form of lung cancer during their life. Although the association of smoking with lung cancer has been convincingly demonstrated, there are at present no ways to predict individual cancer risk beyond smoking history. One alternative way to determine cancer risk would be to develop ways to test for genetic damage in either the target tissue, lung, or in other cells which were exposed to the carcinogens or their metabolites, for instance bladder cells. Since cancer is a disease of the DNA, a generalized measure for DNA damage could be used as a surrogate marker for damage to cancer-associated genes. Our working hypothesis is therefore: that genetic damage in specific sites of the genome can be used as a combined measure for exposure, susceptibility, and efficacy of DNA repair mechanisms. Recently, it has been shown that normal or hyperplastic tissues may already harbor the genetic changes in genes that can be found in overt malignancies. In early studies, these genetic alterations could be demonstrated in bronchial lavages where they were found to precede the onset of lung cancer. Since alterations in cancer-causing genes are directly related to a growth-advantage, the number of cells harboring these alterations may be large enough to detect in small, and partly clonal, cell populations. However, at present there are only two genetic alterations which are known to occur early enough to serve as a marker to predict lung cancer risk: point mutation in the K-ras oncogene and deletion of a putative tumor suppressor gene on the short arm of chromosome 3. Initially, we will employ assays for K-ras point mutations that are sensitive enough to detect a small number of mutated cells that are present in histologically normal lung tissue. We will use these assays to determine the prevalence of K-ras mutations in human lung tissue samples in relation to lung cancer risk, genetic background and exposure. Another area of research will be the investigation of "at risk motifs", or ARMs, which may be at an increased risk for alteration in response to DNA damage. Research in a yeast based mutagenesis system performed at the NIEHS has demonstrated that such sequences directly determine the stability of the DNA in which they occur. The results of these yeast-based model systems could possibly be used to identify novel sequences in the human genome that may exert greater than normal DNA damage after exposure, and could thus serve as additional markers for lung cancer risk. Along these lines, we have explored the possibility that the "Alu" repeated element in human DNA is involved in the inactivation of the p53 tumor suppressor gene, either by driving homologous recombinational events, or by serving as templates to repair DNA breaks by homologous recombination. In the Hs766T human pancreatic cell line, an intragenic deletion in p53 was characterized at the molecular level. We found that in this cell line, p53 exons 2 through 4 are deleted in a complex recombinational event that involves two Alu elements adjacent to these exons, but also one additional Alu element from p53 intron 1. Currently, we are investigating a novel minisatellite marker, MSY1, for cancer-specific changes. This Y-chromosome specific marker was analyzed in a panel of 23 male human cell lines and found to be present in only 14. In two of these 14 cell lines, an aberrant pattern of MSY1 PCR amplification was found and we are currently investigating the nature of these DNA sequences.